CN110946988B - Application of short peptide in preparing pharmaceutical composition for inhibiting or slowing shrimp allergic reaction - Google Patents

Application of short peptide in preparing pharmaceutical composition for inhibiting or slowing shrimp allergic reaction Download PDF

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CN110946988B
CN110946988B CN201811123245.8A CN201811123245A CN110946988B CN 110946988 B CN110946988 B CN 110946988B CN 201811123245 A CN201811123245 A CN 201811123245A CN 110946988 B CN110946988 B CN 110946988B
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short peptide
shrimp
pharmaceutical composition
inhibiting
leu
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CN110946988A (en
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王育民
杜军毅
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Zhenwei Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The invention provides application of a short peptide in preparing a composition for inhibiting or relieving shrimp anaphylactic reaction, which comprises the unglycosylated short peptide as an effective component and can obviously inhibit or relieve symptoms related to the shrimp anaphylactic reaction.

Description

Application of short peptide in preparing pharmaceutical composition for inhibiting or slowing shrimp allergic reaction
Technical Field
The invention relates to a composition for inhibiting or relieving anaphylaxis, in particular to a composition containing short peptide and application of the short peptide in preparing a pharmaceutical composition for inhibiting or relieving anaphylaxis of shrimp.
Background
Food allergy is a state of hypersensitivity caused by acute hyperimmune reaction to foreign, harmless antigens contained in food. Generally, foods which are liable to cause allergy include crustacean seafood (e.g., shrimps), foods containing artificial food additives, legume foods, stone fruit foods, caffeine-containing foods, partial fruits (e.g., mango, strawberry, tomato, orange), alcoholic beverages or foods, other foods (e.g., eggs, milk, shiitake mushrooms, bamboo shoots, agricultural chemical-residue greens), and the like.
Shrimps are widely used in various food materials, dishes and processed foods. In the case of shrimp allergy, it is also associated with genetics, age, and asthma of individuals in addition to shrimp. To avoid shrimp allergy, in addition to preventing the ingestion of shrimp or food containing shrimp components in advance, after shrimp allergy has occurred, appropriate drugs (e.g., antihistamine drugs, steroid drugs, cromolyn sodium and the like) are administered to the shrimp to suppress the shrimp allergy in vivo, depending on the severity of symptoms and the site of onset of the allergy.
However, the above drugs have various side effects to different degrees. For example, common side effects of antihistamines are somnolence, tiredness, inattention, etc., common side effects of steroids are moon face, buffalo back, etc., and common side effect of cromolyn sodium is tingling sensation at the beginning of use.
In view of the above, there is a need to develop new pharmaceutical ingredients that effectively inhibit or slow down food allergy and reduce side effects.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a composition containing a short peptide, which comprises an unglycosylated short peptide as an effective ingredient for inhibiting or alleviating a shrimp allergy in a subject.
Another object of the present invention is to provide a use of a short peptide for preparing a pharmaceutical composition for inhibiting or alleviating a shrimp allergy, which comprises the short peptide without glycosylation.
In accordance with the above object of the present invention, there is provided a short-peptide-containing composition comprising a short peptide as an active ingredient. In this embodiment, the short peptide can be, for example, an aglycosylated short peptide consisting of the amino acid sequence shown in SEQ ID NO. 1, to inhibit or slow the shrimp allergic reaction in the subject.
In an embodiment of the present invention, the composition may be, for example, a food composition or a pharmaceutical composition.
In an embodiment of the present invention, the subject may be, for example, an animal cell or a mammal.
According to another object of the invention, the use of a short peptide for preparing a pharmaceutical composition for inhibiting or alleviating shrimp allergic reactions is provided. In this example, the short peptide is an unglycosylated short peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
In one embodiment of the present invention, the shrimp allergic reaction includes an increase in the expression of histamine, β -hexosaminidase, TNF- α, IL-4, IL-6, IL-13, COX-2 and/or IgE antibodies.
In an embodiment of the present invention, the pharmaceutical composition is administered to a subject, and the subject is an animal cell or a mammal.
In an embodiment of the present invention, the effective dose of the above-mentioned short peptide in the pharmaceutical composition can be, for example, 5.7 μ g/g body weight to 9.6 μ g/g body weight.
The composition containing the short peptide comprises the unglycosylated short peptide as an effective component, can obviously inhibit or slow down the symptoms related to the shrimp anaphylactic reaction, and is further used for preparing the pharmaceutical composition for inhibiting or slowing down the shrimp anaphylactic reaction.
Drawings
The foregoing and other objects, features, and advantages of the invention will be apparent from the following more particular description of the embodiments of the invention, as illustrated in the accompanying drawings in which:
FIG. 1A is a bar graph showing the cell survival rate of a pharmaceutical composition containing a short peptide according to an embodiment of the present invention after administration to a Rat Basophilic Leukemia (RBL) cell line RBL-2H3 (hereinafter, RBL-2H3 mast cell).
[ FIG. 1B ] is a bar graph showing the histamine release rate of a pharmaceutical composition containing a short peptide according to an embodiment of the present invention after administration to RBL-2H3 mast cells.
FIG. 1C shows a bar graph of beta-hexosaminidase activity of a short peptide-containing pharmaceutical composition according to one embodiment of the present invention after administration to RBL-2H3 mast cells.
[ FIG. 1D ] to [ FIG. 1H ] are bar graphs showing the relative content changes of TNF-. Alpha.mRNA (FIG. 1D), IL-4 mRNA (FIG. 1E), IL-6 mRNA (FIG. 1F), IL-13 mRNA (FIG. 1G) and COX-2 mRNA (FIG. 1H), respectively, of the pharmaceutical composition containing a short peptide according to one embodiment of the present invention after administration to RBL-2H3 mast cells.
FIG. 1I shows Western blot analysis of related gene expression of Fc ε RI-dominated signaling pathway after IgE/DNP-induced hypersensitivity of RBL-2H3 mast cells followed by administration of a short peptide-containing pharmaceutical composition according to one embodiment of the present invention.
FIG. 2 is a Western blot analysis of the expression of genes involved in the Fc ε RI-dominated signaling pathway after allergic reaction induced by allergic serum in RBL-2H3 mast cells followed by administration of a short peptide-containing pharmaceutical composition according to one embodiment of the present invention.
FIG. 3 is a dispersion chart showing the change of the relative content of IgE antibodies in mice according to one embodiment of the present invention after they are administered with a short-peptide-containing pharmaceutical composition after they induced an allergic reaction by shrimp extract.
Detailed Description
As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Numerical ranges (e.g., 10% to 11% of A) include upper and lower limits unless otherwise specified (i.e., 10% to 11% A); numerical ranges without lower limits (e.g., less than 0.2% B, or less than 0.2% B) are all meant to indicate that the lower limit may be 0 (i.e., 0% to 0.2%). The words used above are words of description and understanding, rather than words of limitation.
The present invention provides a pharmaceutical composition containing a short peptide, which comprises an unglycosylated short peptide as an effective ingredient for suppressing or alleviating a shrimp allergy in a subject.
As used herein, "shrimp allergy" is a direct correlation with food allergy. The only shrimp allergens currently known are tropomyosin (tropimyosin). After shellfish (shellfish) such as shrimps are eaten, allergens in food (for example, shrimp allergens are tropomyosin, tropomyosin) stimulate the immune system, and various acute allergic symptoms are caused in various parts of the body, such as digestive systems (for example, abdominal pain, nausea, vomiting or diarrhea), skins (for example, red and itchy skins, urticaria, eczema and the like), respiratory systems (for example, running nasal water, sneezing, chest distress, shortness or difficulty in breathing, asthma, cough, red and itchy eyes, swelling and pain of the oropharynx, tingling sensation of the mouth and throat and the like), headache, dizziness, hypotension, even anaphylactic shock (anaphalic shock) and the like.
The results of the present study indicate that the aforementioned "shrimp allergy" is highly correlated with mast cells (mast cells). After eating shellfish seafood such as shrimp, allergen in food interacts with antibody IgE and binds to the surface of cells (e.g., mast cells) having Fc epsilon RI, so that mast cells (mast cells) are degranulated (degranulation), and pro-inflammatory cytokines (TNF-alpha, IL-4, IL-6, IL-13), histamine, beta-hexosaminidase, etc. are released, and via the Fc epsilon RI (receptor for IgE antibody) dominant signal transmission pathway, AKT, ERK, JNK, p38, etc. (e.g., the amount of mRNA expressed and/or the amount of protein phosphorylated) downstream of Fc epsilon RI are activated, and the synthesis of pro-inflammatory cytokines (de novo) is initiated, and the expression of antibody IgE is increased, thereby exacerbating the symptoms of anaphylaxis.
As used herein, "inhibiting or alleviating an allergic reaction in shrimp" refers to the action of directly or indirectly inhibiting or alleviating any of the following conditions caused by an allergic reaction in shrimp, after a subject is administered with the pharmaceutical composition containing the short peptide: various acute allergic symptoms (digestive system, skin, respiratory system, etc.); the content of proinflammatory cytokines (TNF-alpha, IL-4, IL-6, IL-13), histamine, beta-hexosaminidase, etc. in vivo; the expression level of AKT, ERK, JNK, p38 and the like downstream of fceri (e.g., the expression level of mRNA and/or the amount of protein phosphorylation); novel (de novo) synthesis of pro-inflammatory cytokines; and the expression level of antibody IgE. In addition, proinflammatory cytokines or cytokines (cytokines) stimulate, and induce the production of cyclooxygenase-2 (COX-2). Because the pharmaceutical composition contains the short peptide with the specific sequence, the shrimp anaphylactic reaction can be effectively inhibited or slowed down in a consumption inhibition or competitive inhibition mode.
As used herein, the term "short peptide" refers to a short chain polypeptide having a total number of amino acid residues of no more than 40, preferably no more than 37. In one embodiment, the short peptide is an aglycosylated short peptide consisting of the amino acid sequence shown in SEQ ID NO. 1, wherein the amino acid sequence shown in SEQ ID NO. 1 is the sequence referenced to Genbank accession No. NP-002843.2, which is incorporated herein by reference. It is described that, if the total number of amino acid residues in the short peptide exceeds 37, the effective dose of the short peptide thus obtained is difficult to increase because of its large molecular weight. Secondly, if the short peptide does not completely contain the specific sequence shown in SEQ ID NO. 1, the effective amino acid region of the short peptide thus obtained is insufficient, and the effect of inhibiting or alleviating the shrimp allergic reaction in the subsequent application is impaired.
In one embodiment, the non-glycosylated short peptide can be synthesized by any conventional method, such as artificial synthesis, or recombinant protein expression using recombinant genes in an expression system. The method for producing synthetic peptide or recombinant protein is well known to those skilled in the art, and will not be described herein.
It is stated here that the short peptides of the amino acid sequence shown in SEQ ID NO. 1 must be non-glycated. If the short peptides are pre-glycated, they are themselves biologically active and are unable to inhibit or slow shrimp allergic reactions by consumption inhibition or competitive inhibition.
In use, the short peptides can be used for preparing a composition for inhibiting or alleviating shrimp allergic reaction, such as a food composition or a pharmaceutical composition. In one embodiment, the dosage form of the pharmaceutical composition is not limited, and the effective dosage of the short peptide in the pharmaceutical composition can be flexibly adjusted according to the dosage form and the application site. In the case of oral pharmaceutical compositions, the effective dose of the short peptide in the pharmaceutical composition can be, for example, 5.7 μ g/g to 9.6 μ g/g body weight, preferably 6.4 μ g/g to 8.1 μ g/g body weight. In other dosage forms of the pharmaceutical composition, such as an oral nasal spray, the effective dose of the short peptide in the pharmaceutical composition may be adjusted from dosage form to dosage form.
In the above embodiments, the pharmaceutical composition may also optionally comprise a pharmaceutically acceptable carrier. The term pharmaceutically acceptable carrier as used herein refers to a carrier, diluent, filler and/or vehicle which is not an active ingredient per se, but is used to deliver a short peptide as an active ingredient to a subject, or is added to the above-described composition to improve handling or storage properties of the composition, or to allow or facilitate the formation of dosage units of the composition into excipients or any ingredients suitable for pharmaceutical compositions and convenient administration. The aforementioned pharmaceutically acceptable carriers should not destroy the pharmacological activity of the active ingredient and should be non-toxic when delivering a sufficient therapeutic dose of the active ingredient.
The aforementioned suitable pharmaceutically acceptable carriers can be well known to those of ordinary skill in the art of manufacturing pharmaceutical compositions and include, but are not limited to, buffers, diluents, disintegrants, binders, adhesives, humectants, polymers, lubricants, glidants, ingredients added to mask or counteract an unpleasant taste or odor, dyes, fragrances, and ingredients added to improve the appearance of the composition. Specific examples of the aforementioned pharmaceutically acceptable carriers may include, but are not limited to, citrate buffers, phosphate buffers, acetate buffers, bicarbonate buffers, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, magnesium carbonate, talc, gelatin, acacia, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulosic components (such as cellulose esters and cellulose alkyl esters of alkanoic acids), sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerin or powders, polymers (such as polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol), and other pharmaceutically acceptable ingredients.
In one embodiment, the route of administration of the composition containing a short peptide is not particularly limited, and may be oral route or non-oral route (e.g., subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, respiratory inhalation, etc.).
The composition containing the short peptide is proved to have the efficacy of inhibiting or slowing the shrimp allergic reaction of a subject through animal cell or mammal animal experiments. In one example, the aforementioned effects of inhibiting or slowing shrimp allergy in a subject can include, but are not limited to, inhibiting or slowing overexpression of pro-inflammatory cytokines (TNF- α, IL-4, IL-6, IL-13), histamine, β -hexosaminidase, antibody IgE, and the like. In other examples, the aforementioned efficacy of inhibiting or slowing a shrimp allergy in a subject can include, but is not limited to, inhibiting or slowing AKT, ERK, JNK, p38, etc. (e.g., the amount of mRNA expression and/or the amount of protein phosphorylation) downstream of fceri, thereby inhibiting or slowing a shrimp allergy.
The following examples are provided to illustrate the present invention, but not to limit the invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention.
EXAMPLE I preparation of short peptides
In this example, an artificially synthesized short peptide RI37 (having an amino acid sequence shown by SEQ ID NO: 1) was used as the short peptide, and a polypeptide pPTX3 (having an amino acid sequence shown by SEQ ID NO: 2) was used as a control group.
The short peptide RI37 can be synthesized by, for example, koloni International technology Co., ltd. The pPTX3 is a recombinant protein expressed and purified by the present inventors using a conventional prokaryotic expression system (E.coli). The method for producing synthetic peptide or recombinant protein is well known to those skilled in the art, and will not be described herein.
The second embodiment: establishing a cell test model
1. Cell culture
In this example, rat Basophilic Leukemia (RBL) cell line RBL-2H3 (ATCC No. CRL-2256; hereinafter referred to as RBL-2H3 mast cell) was selected and the inhibitory or slowing effect of the short peptide of the first example on mast cell degranulation was evaluated.
The RBL-2H3 mast cells can be cultured by using a special cell culture and passage mode. For example, RBL-2H3 mast cells are cultured in Dulbecco's modified Eagle's medium (DMEM; gibco Co.) cell culture medium containing 10% Fetal Bovine Serum albumin (Fetal Bovine Serum, FBS, which has been inactivated by heat) and antibiotics (50 to 100. Mu.g/mL streptomycin and 50 to 100U/mL penicillin), and cultured at 37 ℃ in 5% carbon dioxide with humidity, which is generally known in the art of the present invention and therefore not described in detail.
MTT assay
MTT is tetrazolium salt (tetrazolium salt), and blue formazan (formazan) crystals are generated from transparent and colorless substances after decomposition by granule glandular dehydrogenase (dehydrogenase) in living cells, and the influence of the short peptide of example one on the survival rate of RBL-2H3 cells of example two is judged by taking the survival rate of cells to which the short peptide of example one is not added as 100%.
First, the RBL-2H3 mast cells of example two were cultured in a 96-well cell culture dish (cell density 2X 10) 4 cells/well), co-cultured with RI37 (300. Mu.g/mL) short peptide of example one at 37 ℃ for 24 hours,After 48 hours or 72 hours, 5mg/mL of MTT [3- (4, 5-dimethylthiozol-2-yl) -2,5-diphenol tetrazolium boss](Sigma) was reacted at 37 ℃ for another 3 hours. The precipitated MTT blue formazan crystals are then dissolved out using DMSO, using a commercially available microplate reader (microplate reader; e.g., iMark) TM Microplate adsorption Reader, bio-Rad) measures the Absorbance of cells at 570nm to assess cell viability.
FIG. 1A is a bar graph showing the cell viability of RBL-2H3 mast cells after administration of the pharmaceutical composition, wherein the vertical axis represents the absorbance at 570nm, the horizontal axis is followed by the addition of short peptides, the horizontal axis is followed by the graph number "ns", the graph number "ns" represents no statistically significant difference (no sign information; ns).
As can be seen from the results shown in fig. 1A, the short peptide RI37 according to the first embodiment of the present invention, which was co-cultured with RBL-2H3 mast cells for 24 hours, 48 hours, or 72 hours, had no significant effect on cell survival rate, indicating no cytotoxicity.
In addition, the short peptide RI37 of the first embodiment of the present invention has no significant effect on the cell number and the mRNA of FcgRI (receptor for IgE antibodies) after being cultured together with RBL-2H3 mast cells (not shown), which means that the short peptide RI has no effect of promoting the increase of the number of RBL-2H3 mast cells and FcgRI.
3. Assessing degranulation of cells
To evaluate degranulation of cells, RBL-2H3 mast cells of example two were first cultured in a 24-well cell culture dish (cell density 2X 10) 5 cells/well), and cell sensitization (cell sensitization) was performed by overnight culture with 0.2. Mu.g/mL of anti-DNP (2, 4-dinonylhydrazine) -IgE antibody. Then, cells were washed with 1-fold PBS (1X PBS). Next, cells were pretreated with short peptide (300 ng/mL) for 30 min at 37 ℃. Then, the polypeptide pPTX3 (300 ng/mL, abbreviated as PTX 3) or 1. Mu.g/mL of DNP-BSA (dissolved in PIPES buffer solution containing 140mM NaCl,5mM KCl,0.6mM MgCl) 2 ,1mM CaCl 2 5.5mM glucose,0.1% BSA and 10mM PIPES, pH 7.4 as controlsGroup) as antigen (Ag), cells were stimulated for 30 min.
Thereafter, cell supernatants were collected and the contents of histamine and the activity of β -hexosaminidase were measured, respectively, as indicators for evaluating cell degranulation.
3.1. Evaluation of the release rate of histamine from cells
The histamine release rate was calculated according to the following conventional method. First, 100. Mu.L of cell supernatant was mixed with 20. Mu.L of 1M NaOH, and then 25. Mu.L of a reaction solution [ containing 1% (w/v) o-phthalaldehyde (o-phthalaldehyde) dissolved in methanol ] was added and reacted at room temperature (generally 10 ℃ C. To 40 ℃ C.) for 4 minutes. Then, 10. Mu.L of 3M HCl was added to the above reaction mixture to terminate the reaction. Then, the fluorescence intensity was measured at an excitation wavelength of 355nm and an emission wavelength of 460nm, and the results are shown in FIG. 1B.
FIG. 1B is a bar graph showing the histamine release rate of RBL-2H3 mast cells administered with the short peptide-containing pharmaceutical composition according to one embodiment of the present invention, wherein the graph symbol "+" below the horizontal axis represents the addition of the short peptide or polypeptide PTX3 during cell culture, the graph symbol "-" represents the absence of the addition of the short peptide or polypeptide PTX3 during cell culture, and the graph symbol "p" represents statistically significant (p < 0.001), and the amount of histamine released by cell lysis buffer (lys buffer) after cell disruption is taken as a 100% basis.
From the results in FIG. 1B, it is clear that IgE can activate RBL-2H3 mast cells, cause degranulation and release histamine. Stimulation of IgE sensitized mast cells with antigen (PTX 3) may exacerbate the degranulation reaction and release more histamine. However, the pretreatment of IgE-sensitized RBL-2H3 mast cells with the short peptide RI37 of the first embodiment of the present invention before stimulation and degranulation with antigen (i.e., PTX 3) can significantly inhibit or slow down degranulation of IgE/Ag-activated RBL-2H3 mast cells, and reduce the release rate of histamine, with statistically significant differences.
3.2. Assessing beta-hexosaminidase activity of cells
When the activity of beta-hexosaminidase was evaluated, the same principle was appliedThe method described in point 3, except that 25. Mu.L of the above collected cell supernatant was mixed with an equal volume of 5mM matrix solution containing 5mM p-nitrophenyl N-acetyl- β -D-glucopyranoside (p-NAG) dissolved in 0.2M sodium citrate buffer solution, pH 4.5, and then 25. Mu.L of a reaction solution [ containing 1% (w/v) o-phthalaldehyde (o-phthaloaldehyde) dissolved in methanol ] was added and reacted at 37 ℃ for 1.5 hours. Then, 200. Mu.L of a stop solution (containing 0.1M Na) was added to the above reaction mixture 2 CO 3 /NaHCO 3 pH 10.0) to terminate the reaction. Then, the absorbance of the cells at 405nm was measured to determine the activity of β -hexosaminidase, and the results are shown in fig. 1C.
Referring to FIG. 1C, a bar graph of beta-hexosaminidase activity of a pharmaceutical composition containing a short peptide according to one embodiment of the present invention after administration to RBL-2H3 mast cells is shown, wherein the vertical axis represents the absorbance of the cells at 405nm, the graph number below the horizontal axis represents the addition of the short peptide or polypeptide PTX3 during cell culture, the graph number represents the absence of the addition of the short peptide or polypeptide PTX3 during cell culture, the graph number represents data having statistical significance (p < 0.01), and the graph number represents data having statistical significance (p < 0.001).
As shown in FIG. 1C, igE activates RBL-2H3 mast cells, causing degranulation and release of β -hexosaminidase. Stimulation of IgE sensitized mast cells with antigen (PTX 3) exacerbates the degranulation reaction and releases more β -hexosaminidase. However, igE-sensitized RBL-2H3 mast cells treated with RI37, a short peptide according to the first embodiment of the present invention, before stimulation and degranulation by antigen (i.e., PTX 3), can significantly inhibit or slow down degranulation of IgE/Ag-activated RBL-2H3 mast cells, and reduce β -hexosaminidase activity, and the difference is statistically significant.
4. Assessing gene expression of proinflammatory cytokines in cells
Total RNA from the IgE-sensitized mast cells stimulated with the antigen (PTX 3) at point 3 above was extracted using a commercially available extraction kit (e.g., TRIsure RNA extraction reagent, invitrogen). Subsequently, the total RNA is subjected to a reverse transcription reaction using a commercially available reverse transcriptase (e.g., superScript III, invitrogen) to synthesize cDNA. Then, quantitative PCR (qPCR) of each target gene was performed in a commercially available Real-Time PCR System (e.g., CFX connect Real-Time PCR System, BIO-RAD) using a commercially available fluorescent Real-Time quantitative qPCR reaction kit (e.g., KAPA SYBR FAST qPCR Master Mix, life Technologies Corporation and Kapa Biosystems Inc.) and primers listed in SEQ ID NOs: 3-14, and the results are shown in FIGS. 1D to 1H.
Please refer to FIGS. 1D-1H, which respectively show the bar graphs of the relative content changes of TNF- α mRNA (FIG. 1D), IL-4 mRNA (FIG. 1E), IL-6 mRNA (FIG. 1F), IL-13 mRNA (FIG. 1G), and COX-2 mRNA (FIG. 1H) after administration of the pharmaceutical composition containing short peptides to RBL-2H3 mast cells according to one embodiment of the present invention, wherein the content of β -actin mRNA is used as an internal control group (not shown). The graph numbers below the horizontal axis of FIGS. 1D to 1H represent the addition of the short peptide or polypeptide PTX3 during cell culture, the graph numbers represent the absence of the addition of the short peptide or polypeptide PTX3 during cell culture, the graph numbers represent data with statistical significance (p < 0.05), the graph numbers represent data with statistical significance (p < 0.01), and the graph numbers represent data with statistical significance (p < 0.001), and the mRNA content of β -actin (β -actin) in each group is used as an internal control group.
As is clear from the results shown in FIGS. 1D to 1H, the expression levels of TNF-. Alpha.IL-4, IL-6 mRNA, IL-13 and COX-2 mRNA were significantly increased by stimulating IgE-sensitized mast cells with the antigen (PTX 3). However, after pretreatment of IgE-sensitized RBL-2H3 mast cells with the short peptide RI37 of the first embodiment of the present invention, the antigen (i.e., PTX 3) is used to stimulate the IgE-sensitized mast cells, so that the expression levels of TNF-alpha, IL-4, IL-6 mRNA, IL-13 and COX-2 mRNA can be significantly inhibited or slowed down, and the difference has statistically significant difference.
5. Assessing Gene expression (I) of intracellular signalling pathways
In assessing gene expression in intracellular signaling pathway, the procedure of point 3 above was followed in principle, except that after pretreatment of cells at 37 ℃ for 30 minutes with different concentrations of short peptide (150 ng/mL or 300 ng/mL), the cells were stimulated with the polypeptides PTX3, DNP-IgE/DNP. Thereafter, all cell lysates were collected and subjected to Western blot analysis (Western blotting analysis).
Western blot analysis can be performed in a conventional manner. First, the protein of the cells was extracted using a modified radioimmunoprecipitation assay (modified RIPA) buffer solution. The RIPA buffer solution of the aforementioned modified formulation contained 50mM Tris-HCl [ pH 7.4], 150mM sodium chloride, 1mM ethylenediamine tetraacetate (EDTA), 1% ethylphenylpolyethylene glycol (octoxyphenylthionol, nonidet P40, NP40), 0.25% sodium deoxycholate (sodium deoxycholate), 1mM Dithiothreitol (DTT), 1mM Phenylmethylsulfonylfluoride (PMSF), 1. Mu.g/mL aprotinin (aprotinin), and 1. Mu.g/mL leupeptin (leupeptin).
After the cell lysate obtained as described above was electrophoresed on SDS-PAGE, cell proteins were blotted from the SDS-PAGE gel onto a PVDF (polyvinylidene difluoride membrane) membrane
Figure GDA0003867524060000171
The primary antibodies were allowed to act overnight at 4 ℃ using primary antibodies including an anti-p 84 antibody (model: GTX70220, geneTex), an anti-phosphorylated AKT (p-AKT) antibody (model: GTX121937, geneTex), an anti-AKT antibody (model: GTX128414, geneTex), an anti-p-ERK antibody (model: 4377, cell Signaling), an anti-ERK antibody (model: 9102, cell Signaling), an anti-p 38 antibody (model: 9211, cell Signaling), an anti-p 38 antibody (model: 9212, cell Signaling), an anti-p-JNK antibody (model: 4668, cell Signaling), and an anti-p-JNK antibody (model: 9252, cell Signaling). Then, the cells were incubated for 1.5 hours at room temperature with a secondary antibody that binds peroxidase (peroxidase). Thereafter, the expression level of the protein was measured using a commercially available color kit (e.g., ECL kit, perkinElmer) and the protein expression level of p84 was used as an internal control group.
Protein extraction and western blot analysis are well known to those skilled in the art, and will not be described in detail herein.
Please refer to fig. 1I, which shows a western blot analysis of related gene expression of fceri-dominated signaling pathway after IgE/DNP-induced hypersensitivity reaction of RBL-2H3 mast cells followed by administration of a pharmaceutical composition containing short peptides, respectively, according to one embodiment of the present invention. In FIG. 1I, the graph "plus" represents the addition of the short peptide or PTX3 during cell culture, the graph "minus" represents the absence of the addition of the short peptide or PTX3 during cell culture, and the triangle represents the incremental increase in the addition of the specific short peptide.
From the results shown in FIG. 1I, it was found that the stimulation of IgE/DNP-sensitized mast cells with antigen (PTX 3) significantly increased the amount of proteins such as AKT, ERK1/ERK2, p38, and JNK1/JNK2 phosphorylated to p-AKT, p-ERK1/ERK2, p-p38, and p-JNK1/JNK2, and aggravated the allergic reaction (agravation). However, after the IgE/DNP sensitized RBL-2H3 mast cells are pretreated by the short peptide RI37 of the embodiment I, and then the antigen (PTX 3) is used for stimulating the IgE/DNP sensitized mast cells, the phosphorylation of proteins such as AKT, ERK1/ERK2, p38, JNK1/JNK2 and the like into p-AKT, p-ERK1/ERK2, p-p38, p-JNK1/JNK2 can be obviously inhibited or slowed down.
6. Assessment of Gene expression in intracellular signalling pathways (II)
In order to evaluate the gene expression in the intracellular signaling pathway, the procedure of the above 3 rd point was basically the same, except that the cells were pretreated with short peptides (150 ng/mL or 300 ng/mL) at different concentrations at 37 ℃ for 30 minutes, and then the cells were stimulated with the serum of mice sensitized with the shrimp extract (10. Mu.L/mL of serum was added). Thereafter, all cell lysates were collected, subjected to the same western blot analysis as at point 5, and the protein expression level of p84 was used as an internal control group.
The above shrimp extract is prepared by the following method. First, the shrimp is dehulled and the meat portion is taken and added to a fixed proportion of redistilled water (ddH) 2 O; also known as secondary water) (shrimp meat: secondary water = 1. Centrifuging the meat paste at 8000rpm for 30 min, collecting supernatant, and freeze drying to obtain the final productA shrimp extract. The resulting shrimp extract was injected intraperitoneally (i.p.) to sensitize (sensitization) and challenge (challenge) mice to induce shrimp allergic reactions in the mice. For sensitization and stimulation of mice, see point 7. Then, the allergic serum of the shrimp-allergic mouse was taken as an antigen to stimulate RBL-2H3 mast cells.
Please refer to fig. 2, which is a western blot analysis of the expression of genes involved in the Fc epsilon RI-dominated signaling pathway after allergic reaction induced by allergic serum in RBL-2H3 mast cells followed by administration of a pharmaceutical composition comprising a short peptide according to an embodiment of the present invention. In FIG. 2, the graph "plus" represents the addition of the short peptide or PTX3 during cell culture, the graph "minus" represents the absence of the addition of the short peptide or PTX3 during cell culture, and the triangle represents the increasing addition amount of the specific short peptide.
As is clear from the results shown in FIG. 2, the stimulation of IgE-sensitized mast cells with the allergy serum significantly increased the protein expression levels of p-AKT, p-ERK1/ERK2, p-p38, and p-JNK1/JNK 2. However, after the IgE-sensitized RBL-2H3 mast cells are pretreated by the short peptide RI37 of the embodiment of the invention, the allergic serum is used for stimulating the IgE-sensitized mast cells, so that the protein expression levels of p-AKT, p-ERK1/ERK2, p-p38 and p-JNK1/JNK2 can be obviously inhibited or slowed down.
7. Assessment of IgE content in vivo
This example evaluates the effect of short peptide RI37 in inhibiting or slowing shrimp allergic reactions in female Balb/c mice (6 mice per group) of 3 to 4 weeks of age by intraperitoneal (i.p.) injection of antigen (PBS or shrimp extract) to elicit shrimp allergic reactions. The body weight of the female Balb/c mice aged 3 to 4 weeks is 10.4 g/mouse to 17.4 g/mouse on average.
Control groups (groups 1 and 2) mice were injected intraperitoneally with PBS and aluminum-containing adjuvant [ aluminum hydroxide; al (OH) 3 ](in PBS) and mice were injected intraperitoneally with PBS on day 30. Shrimp allergic groups (groups 3 and 4) mice were intraperitoneally injected with 1-fold dose of shrimp extract on days 1, 4, 7, 10, 13, 16, 19, and 22 with aluminum-containing adjuvant [ aluminum hydroxide; al (OH) 3 ](in PBS) sensitized (Sensitizati)on) and mice were challenged (challenge) with 5 x intraperitoneal injection of shrimp extract on day 30 to elicit a shrimp allergic reaction. The amount of the shrimp extract in the above 1-fold dose was 0.5mg/g body weight.
Short peptide RI 37-treated groups (groups 2 and 4) mice were injected intraperitoneally with short peptide RI37 (dissolved in PBS, group 2), or short peptide RI37 and shrimp extract (dissolved in PBS, group 4) on days 19, 22, and 30.
Whole blood from all mice was collected from the mouse eye sinuses (orbital sinuses) 2 hours after stimulation on day 30 to assess IgE content in the mice.
The whole blood obtained as described above was allowed to stand (i.e., without stirring) at room temperature for 30 minutes to coagulate the whole blood. Next, the mixture was centrifuged at 2000g for 10 minutes in a low temperature centrifuge (dried centrifuge). Then, the content of IgE (1.
Please refer to fig. 3, which is a dispersion diagram showing the change of the relative content of IgE antibodies in mice after they are administered with the pharmaceutical composition containing short peptides after the shrimp extract induces the allergic reaction according to one embodiment of the present invention. In FIG. 3, the vertical axis represents the absorbance at a wavelength of 450nm, the horizontal axis represents groups 1 to 4 from left to right, groups 1 and 2 represent control groups, groups 3 and 4 represent shrimp allergy groups, groups 2 and 4 are treated with short peptides, the graph number "+" represents the addition of short peptides during cell culture, the graph number "-" represents the absence of short peptides during cell culture, the graph number "Pinggao" represents data with statistical significance (p < 0.01), the graph number "Pinggao" represents statistical significance (p < 0.001), and the graph number "ns" represents no significant difference (no signifiancan; ns).
As can be seen from the results of groups 1 and 2 of fig. 3, the content change of IgE antibody was not statistically significantly different between the mice stimulated with the short peptide RI 37. As is clear from the results of groups 1 and 3 in fig. 3, the content of IgE antibody was significantly increased by the stimulation of mice with shrimp extract. However, as shown in the results of group 4 of fig. 3, the shrimp allergic mice treated with the short peptide RI37 of the first embodiment of the present invention at days 19, 22 and 30 significantly inhibited or slowed the content of IgE antibodies, and the difference was statistically significant, so as to achieve the purpose of inhibiting or slowing the shrimp allergic reaction.
In addition, the short peptide RI37 is used as an effective component, and in vitro and in vivo tests prove that the short peptide RI can really inhibit or slow the shrimp anaphylactic reaction and can be potentially applied to the preparation of food compositions or pharmaceutical compositions for inhibiting or slowing the shrimp anaphylactic reaction. In the application, the food composition or the pharmaceutical composition of the present invention may be administered to prevent, inhibit or alleviate the shrimp allergy before the mast cells caused by the shrimp allergy degranulation or within 30 minutes after the shrimp itself or the food containing the shrimp components is taken.
In summary, although the present invention is exemplified by a short peptide with a specific sequence, a specific dosage form, a specific subject, a specific administration mode, or a specific evaluation mode, the composition containing a short peptide of the present invention and the use thereof for inhibiting or alleviating shrimp allergy are described, but it is known to one of ordinary skill in the art that the present invention is not limited thereto, and the present invention may be carried out using other dosage forms, other subjects, other administration modes, or other evaluation modes without departing from the spirit and scope of the present invention.
As can be seen from the above examples, the composition containing short peptides of the present invention has an advantage in that it contains non-glycosylated short peptides as an active ingredient, can significantly inhibit or alleviate symptoms associated with shrimp allergic reactions, and can be used for preparing a pharmaceutical composition for inhibiting or alleviating shrimp allergic reactions.
While the present invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (6)

1. The use of a short peptide for preparing a pharmaceutical composition for inhibiting or alleviating shrimp allergic reactions, wherein the short peptide is an unglycosylated short peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
2. The use of the oligopeptide according to claim 1 for preparing a pharmaceutical composition for inhibiting or alleviating the allergic reaction of shrimps, wherein the pharmaceutical composition is in the form of an oral composition.
3. The use of the oligopeptide according to claim 1 for preparing a pharmaceutical composition for inhibiting or alleviating a shrimp allergy, wherein the shrimp allergy comprises an increase in the expression of histamine, β -hexosaminidase, TNF- α, IL-4, IL-6, IL-13, C OX-2 and/or IgE antibodies.
4. The use of the oligopeptide according to claim 1 for preparing a pharmaceutical composition for inhibiting or alleviating the allergic reaction in shrimp, wherein the pharmaceutical composition is administered to a subject, and the subject is an animal cell or a mammal.
5. The use of the oligopeptide according to claim 4 for preparing a pharmaceutical composition for inhibiting or alleviating the allergic reaction in shrimps, wherein the effective dose of the oligopeptide in the pharmaceutical composition is 5.7 μ g/g to 9.6 μ g/g of body weight.
6. The use of the short peptide according to claim 4 for preparing a pharmaceutical composition for inhibiting or alleviating shrimp allergy, wherein the effective dose of the short peptide in the pharmaceutical composition is 6.4 μ g/g body weight to 8.1 μ g/g body weight.
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